From chicken genomic DNA. The PCR item was cloned in to the pcDNA3.1 vector in the HindIII and XhoI restriction internet sites to produce the chicken miR-33 over-expression vector pcDNA3.1-miR-33. A negative manage vector pcDNA3.1-NC-miRNA was constructed by inserting into pcDNA3.1 a sequence that had no predicted target web page within the chicken FTO 39UTR. The chicken FTO 39UTR encompassing the predicted miR-33 binding website was amplified by PCR and directionally inserted downstream on the luciferase expression cassette on the pMIR-reporter vector in the SacI and HindIII web sites to construct the pMIR-FTO reporter vector. Point mutations inside the seed area of the predicted miR-33 binding sequence inside the 39UTR of chicken FTO have been generated making use of overlap-extension PCR, plus the resulting plasmid was named pMIR-FTOmut. All constructs have been confirmed by sequencing and ready to lower endotoxin by utilizing the PureLinkTM HiPure Plasmid Filter Purification Kits. RNA Isolation and Real-time qRT-PCR Arbor Acres industrial chickens had been employed in the present study. Several tissues were collected from 4-week-old chickens and liver samples had been taken from 0, 1, two, 3, four, 5, 6 and 7-week-old Primer name ggamiR33 ggaFTO ggaFTOm ggaFTO ggasrebp2 b-actin Primer sequences F/R cccaagcttCTCCATTTCAGGCAGCATCG/ccgctcgagCCAAATCCCTTTTCCCCATC F/R cgagctcTCAGTAGGTAGGATATCAGG/cccaagcttATCCATGGGCTACAAGGTCA F/R GTGCTTCATTCGAAATTCTATTGGTTTCCACC/GGTGGAAACCAATAGAATTTCGAATGAAGCAC F/R TAGTGATTGGAACCTGAAGG/CATCAAGCATCAAGTAGAGG F/R AGCCTCAGATCATCAAGACG/TTCCATTGCTCCCAACAAGG F/R CACGGTATTGTCACCAACTG/ACAGCCTGGATGGCTACATA Solutions length/bp 350 288 288 128 153 200 Tm 58 58 58 58 58 58 Goal Cloning Cloning Cloning qRT-PCR qRT-PCR qRT-PCR doi:ten.1371/journal.pone.0091236.t001 two Expression of miR-33 Targets FTO Gene Transfection of Chicken Hepatocytes Key chicken hepatocytes have been cultured in 12-well plates for approximately 24 h just before transfection. Chicken hepatocytes were transfected with 80 nM miRCURY LNA-anti-miR-33 or LNA scramble control utilizing X-tremeGENE HP DNA transfection reagent. The expression of miR-33 and FTO mRNA was detected 48 h post-transfection. Culture and Transfection of C2C12 Cells C2C12 cells had been obtained from Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. They had been 18334597 maintained in Dulbecco’s Modified Eagle’s three Expression of miR-33 Targets FTO Gene Human ortholog Representative of target gene transcript Gene name Conserved sites 8mer 7mer+m8 7mer+1A Total context+score ABCA1 CROT NAA30 GRB10 ZNF281 NPC1 VCAN ADCYAP1 GLRA1 SLC12A5 IGF1 SCN8A MRPS25 PIM3 CPT1A PRKCE ICK ABHD2 FGF7 RAP2A RMND5A HIPK2 AKAP2 PALM2-AKAP2 GAS1 PCDH18 TPM3 DDX3X ZMIZ1 UBE2V2 PD-168393 web NAP1L4 SIK1 KIAA1409 GRIA3 NM_005502 NM_001143935 NM_001011713 NM_001001549 NM_012482 NM_000271 NM_001126336 NM_001099733 NM_000171 NM_001134771 NM_000618 NM_001177984 NM_022497 NM_001001852 NM_001876 NM_005400 NM_014920 NM_007011 NM_002009 NM_021033 NM_022780 NM_001113239 NM_001004065 NM_007203 NM_002048 NM_019035 NM_001043351 NM_001193416 NM_020338 NM_003350 NM_005969 NM_173354 NM_020818 NM_000828 ATP-binding cassette, sub-family A, member 1 carnitine O-octanoyltransferase N-acetyltransferase 30, NatC catalytic subunit growth aspect receptor-bound protein ten zinc finger protein 281 Niemann-Pick disease, type C1 versican adenylate cyclase GSK -3203591 site activating polypeptide 1 glycine receptor, alpha 1 solute carrier family members 12, member five insulin-like growth aspect 1 sodium chann.From chicken genomic DNA. The PCR item was cloned in to the pcDNA3.1 vector at the HindIII and XhoI restriction web-sites to create the chicken miR-33 over-expression vector pcDNA3.1-miR-33. A adverse control vector pcDNA3.1-NC-miRNA was constructed by inserting into pcDNA3.1 a sequence that had no predicted target site within the chicken FTO 39UTR. The chicken FTO 39UTR encompassing the predicted miR-33 binding web page was amplified by PCR and directionally inserted downstream in the luciferase expression cassette from the pMIR-reporter vector at the SacI and HindIII web sites to construct the pMIR-FTO reporter vector. Point mutations inside the seed region in the predicted miR-33 binding sequence within the 39UTR of chicken FTO had been generated applying overlap-extension PCR, along with the resulting plasmid was named pMIR-FTOmut. All constructs had been confirmed by sequencing and prepared to cut down endotoxin by using the PureLinkTM HiPure Plasmid Filter Purification Kits. RNA Isolation and Real-time qRT-PCR Arbor Acres commercial chickens had been employed in the present study. Numerous tissues were collected from 4-week-old chickens and liver samples were taken from 0, 1, 2, three, four, 5, six and 7-week-old Primer name ggamiR33 ggaFTO ggaFTOm ggaFTO ggasrebp2 b-actin Primer sequences F/R cccaagcttCTCCATTTCAGGCAGCATCG/ccgctcgagCCAAATCCCTTTTCCCCATC F/R cgagctcTCAGTAGGTAGGATATCAGG/cccaagcttATCCATGGGCTACAAGGTCA F/R GTGCTTCATTCGAAATTCTATTGGTTTCCACC/GGTGGAAACCAATAGAATTTCGAATGAAGCAC F/R TAGTGATTGGAACCTGAAGG/CATCAAGCATCAAGTAGAGG F/R AGCCTCAGATCATCAAGACG/TTCCATTGCTCCCAACAAGG F/R CACGGTATTGTCACCAACTG/ACAGCCTGGATGGCTACATA Solutions length/bp 350 288 288 128 153 200 Tm 58 58 58 58 58 58 Goal Cloning Cloning Cloning qRT-PCR qRT-PCR qRT-PCR doi:10.1371/journal.pone.0091236.t001 2 Expression of miR-33 Targets FTO Gene Transfection of Chicken Hepatocytes Main chicken hepatocytes were cultured in 12-well plates for around 24 h ahead of transfection. Chicken hepatocytes have been transfected with 80 nM miRCURY LNA-anti-miR-33 or LNA scramble manage utilizing X-tremeGENE HP DNA transfection reagent. The expression of miR-33 and FTO mRNA was detected 48 h post-transfection. Culture and Transfection of C2C12 Cells C2C12 cells were obtained from Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. They had been 18334597 maintained in Dulbecco’s Modified Eagle’s 3 Expression of miR-33 Targets FTO Gene Human ortholog Representative of target gene transcript Gene name Conserved sites 8mer 7mer+m8 7mer+1A Total context+score ABCA1 CROT NAA30 GRB10 ZNF281 NPC1 VCAN ADCYAP1 GLRA1 SLC12A5 IGF1 SCN8A MRPS25 PIM3 CPT1A PRKCE ICK ABHD2 FGF7 RAP2A RMND5A HIPK2 AKAP2 PALM2-AKAP2 GAS1 PCDH18 TPM3 DDX3X ZMIZ1 UBE2V2 NAP1L4 SIK1 KIAA1409 GRIA3 NM_005502 NM_001143935 NM_001011713 NM_001001549 NM_012482 NM_000271 NM_001126336 NM_001099733 NM_000171 NM_001134771 NM_000618 NM_001177984 NM_022497 NM_001001852 NM_001876 NM_005400 NM_014920 NM_007011 NM_002009 NM_021033 NM_022780 NM_001113239 NM_001004065 NM_007203 NM_002048 NM_019035 NM_001043351 NM_001193416 NM_020338 NM_003350 NM_005969 NM_173354 NM_020818 NM_000828 ATP-binding cassette, sub-family A, member 1 carnitine O-octanoyltransferase N-acetyltransferase 30, NatC catalytic subunit growth aspect receptor-bound protein 10 zinc finger protein 281 Niemann-Pick disease, variety C1 versican adenylate cyclase activating polypeptide 1 glycine receptor, alpha 1 solute carrier household 12, member 5 insulin-like growth element 1 sodium chann.
